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David Duffy, Ulf Landegren, Antoine van Oijen, and Menno Prins crash through the concentration barrier and investigate the ultimate limit of analytical sensitivity.

Being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour‐reactive lymphocytes. We investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour‐specific proliferation and TCR Vβ repertoires. During tumour surgery, sentinel lymph nodes were identified by peri‐tumoural injection of blue dye. Fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single‐cell suspensions were prepared. Cells were assayed for reactivity against autologous tumour extract in [3H]‐thymidine incorporation assays and characterized by flow cytometry. Parallel analyses of the expression of Vβ gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. Probes were reacted with cDNA prepared from magnetically separated CD4+ cells, and the TCR repertoire was determined by hybridizing the products to oligonucleotide microarrays. Dose‐dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. Common clonal expansions were detected among tumour‐infiltrating lymphocytes and in sentinel lymph nodes. Nonsentinel lymph nodes displayed a divergent TCR Vβ repertoire. These results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. Identification of sentinel lymph nodes makes it possible to obtain tumour‐reactive lymphocytes for use in adoptive immunotherapy.